Industrial production of recombinant proteins covers a wide area of developments and applications. The yield of recombinant protein per cell or per L of culture medium is an important asset for the development of improved production systems.
Current methods of expressing genes in mammalian or insect cells for the industrial production of recombinant proteins, monoclonals or vaccines include the use of stable cell lines or the transfection of producer cells using vectors, such as those which are derived from adenoviruses, sindbis viruses, or baculoviruses. Other methods for introduction of an exogenous gene in a mammalian cell or insect cell include direct injection of DNA, the use of ligand-DNA conjugates, the use of adenovirus-ligand-DNA conjugates, calcium phosphate precipitation, and methods which utilize a liposome- or polycation-DNA complex.
In nature, baculoviruses are double-stranded DNA-containing viruses that infect a variety of different insect species. The family of baculoviruses can be divided in two genera, one of which are the nucleopolyhedroviruses. The nucleopolyhedroviruses induce the formation of paracrystalline occlusion bodies in the nuclei of infected host cells. These occlusion bodies are composed primarily of a single viral protein, polyhedrin, which is expressed at very high levels. The polyhedrin gene has been cloned and sequenced and its unique features have provided the basis for the development of a series of baculovirus expression vectors (Summers, M. D. and Smith, G. E., TAES Bull. 1555 (1987); Luckow, V. A. and Summers, M. D., Biotechnology 6:47-55 (1988); Miller, L. K., Ann. Rev. Microbiol. 42:177-179 (1988); U.S. Pat. No. 4,745,051, G. E. Smith and M. D. Summers (Filed May 27, 1983; Issued May 17, 1988)).
The baculovirus-expression system used in conjunction with insect cells has become well-established for the production of proteins, due to its advantages in versatility and speed. In the baculovirus-expression system, a recombinant baculoviral vector is used to introduce a gene of interest into insect cells. Infection of the insect cells results in replication of the recombinant baculovirus vector genome, thereby increasing the number of genetic templates that encode the gene of interest and increasing the level of recombinant protein expression.
Baculovirus-mediated protein expression provides correct folding of recombinant proteins as well as disulfide-bond formation, oligomerization and other important post-translational modifications that provide proper biological activity and function. Indeed, protein folding and post-translational processing of an eukaryotic protein in insect-cells is quite comparable to mammalian cell lines. Furthermore, insect cells can be grown on serum free media which is an advantage in terms of costs as well as of biosafety. Another advantage of baculovirus-mediated protein expression is that baculoviruses only infect Lepidopteran insects, thereby being noninfectious for vertebrates and relatively safe genetic manipulation agents. In addition, the baculovirus-expression system is known to be a technology platform that results in high protein expression levels in insect cells. A disadvantage of the baculovirus expression system is that infection of the producer cell (insect cell) is lethal to that cell within a few days hampering continuous production of the recombinant protein of interest.
Zeng et al. (2007, Stem Cells 25: 1055-1061) disclose a baculovirus expression construct comprising a gene of interest as well as an AAV rep78/68 gene and AAV ITR sequences for integration of the construct into the AAVS1 site in the human genome of human embryonic stem cells.
Sollerbrant et al. (2001, J. Gen. Virol. 82: 2051-2060) disclose mammalian HEK293 cells transfected with separate baculovirus constructs comprising (i) a reporter gene flanked by AAV ITR sequences, (ii) an AAV rep gene, and (iii) an AAV cap gene, respectively, for production of rAAV vectors.
There is however, still a need in the art for increased production levels of gene products of interest in cells such as insect cells.